In normal cell, cell division and its pause occur orderly in accordance with cell cycle, while cancer cell is, on the contrary, characterized by its disorderedness. For this reason abnormality of control mechanism in cell cycle is supposed to have direct relation with oncogenesis or malignant alteration of cancer. Cell cycle of mammalian cell is regulated by serine/threonine kinase called cyclin-dependent kinase (hereinafter abbreviated as to Cdk) family, and Cdk needs to form a complex with the regulatory subunit called cyclin in order to express its activity. Cyclin itself also have a family, and each Cdk molecule is considered to regulate the progression in certain cell cycle by forming a complex with specific cyclin molecule which is expressed at the corresponding stage of the cell cycle. For example, in combination with Cdk4 or Cdk6, D type cyclin regulates the progression of G1 phase, cyclin E-Cdk2 regulates G1/S boundary, cyclin A-Cdk2 regulates the progression of S phase, and cyclin B-cdc2 regulates the progression of G2/M. In addition, three sub-types, D1, D2 and D3 are known as D type cyclin, and moreover the activity of Cdk is considered to be controlled not only by combination with cyclin but also by phosphorylation/dephosphorylation of Cdk molecule, degradation of cyclin molecule and binding with Cdk inhibitor proteins (Advanced Cancer Research, Vol. 66, pp 181-212(1995); Current Opinion in Cell Biology, Vol. 7, pp773-780(1995): Nature, Vol.374, pp131-134 (1995)).
Cdk inhibitor proteins in mammalian cell are classified roughly into two kinds, Cip/Kip family and INK4 family due to the differences in structure and behavior. Compared to that the former inhibits cyclin-Cdk complex widely, the latter inhibits cyclin-Cdk complex specifically, as a result of binding with Cdk4 or Cdk6 (Nature, vol. 366, pp704-707(1993); Molecular and Cellular Biology, vol.15, pp2627-2681(1995): Genes and Development, vol.9, pp1149-1163 (1995)).
For example, P21(Sdi1/Cip1/Waf1) is nominated for a representative example of the former and its expression is induced by cancer repressor gene product, p53.
On the other hand, for example, p16 (INK4a/MTS1/CDK4I/CDKN2) is one of the Cdk inhibitor protein which belongs to the latter. Human P16 gene is encoded on the chromosome 9p21. Abnormalities of this locus are detected with a high frequency in human cancer cell. Actually many reports according to the deletion and mutation of the p16 gene are made in clinical field. And high-frequency of tumorigenesis in p16 knockout-mouse has been reported (Nature Genetics, vol.8, pp27-32(1994); Trends in Genetics, vol.11, pp136-140(1995); Cell, vol.85, pp27-37(1996)).
Each Cdk controls the progression of cell cycle by phosphorylation of a certain target protein at the specific phase of cell cycle, and, above all, retinoblastoma (RB) protein is considered to be the most important target protein. RB protein is the protein which plays an important role in progression from G1 phase to S phase and is rapidly phosphorylated during the term from late G1 phase to initial S phase. This phosphorylation is considered to be carried out by cyclin D-Cdk4/Cdk6 complex followed by cyclin E-Cdk2 complex during the progression of cell cycle. The complex composed of hypophosphorylated RB and transcription factor E2F at early G1 phase dissociates when RB protein becomes hyperphosphorylated. As a result, E2F will be the transcriptional activator, and at the same time, the suppression of the promoter activity by RB-E2F complex will be removed, thus leading to the activity of E2F dependent transcription. At present, the Cdk-RB pathway, which consists of E2F, its suppressor RB protein, Cdk4/Cdk6 which repressively regulates the function of RB protein, Cdk inhibitor protein which controls the kinase activity of Cdk4/Cdk6 and D-type cyclin is thought to be the important mechanism to regulate the progression of GI phase to S phase (Cell, vol.58, pp1097-1105(1989); Cell, vol.65, pp1053-1061(1991); Oncogene, vol.7, pp1067-1074(1992); Current Opinion in Cell Biology, Vol. 8, pp805-814(1996); Molecular and Cellular Biology, vol. 18, pp753-761(1998)). In fact, E2F-binding DNA sequence is located upstream of the sequence related to cell growth relative genes, and in several genes among them the transcription is reported to be activated during the term from late G1 phase to initial S phase(The EMBO Journal, vol.9, pp 2179-2184,(1990); Molecular and Cellular Biology, vol. 13, pp1610-1618 (1993)).
Abnormalities of any factors which intervene in Cdk-RB route, for example, deletion of functional p16, high-expression of cyclin D1, high-expression of Cdk4 and deletion of functional RB protein are found in high frequency in human cancer (Science, vol.254, pp1138-1146(1991); Cancer Research, Vol. 53, pp5535-5541 (1993); Current Opinion in Cell Biology, Vol. 8, pp805-814(1996). All of these promote the progression from G1 phase to S phase abnormally and it is obvious that this route plays an important role in malignant alteration or abnormal growth of cancer cells.
A series of chromone derivatives, for example, by flavopiridol are known as known compounds which have an inhibitory activity on Cdk family (WO 97/16447, 98/13344).
As the prior arts which are structurally similar to the compounds according to this invention, for example, Japanese patent publication Laid open No. 10-502630 (reference A), WO99/46260 (reference B), WO99/46264 (reference C), WO98/13368 (reference D), WO99/50254 (reference E), Japanese patent publication Laid open No. 11-149982(reference F) and Japanese patent publication Laid open No. 04-182471(reference G) are nominated.
In reference A, substituted-2(1H)-pyridopyrazinone derivatives are disclosed. However, such compounds described in reference A are characterized in that they have a substituent other than hydrogen atom at position 1 of the pyrazinone ring, whereas the compounds according to the present invention are characterized in that the substituent at position 1 of the pyrazinone ring is fixed to hydrogen atom, so that the compounds of the present invention are completely different in chemical structure. Moreover, the use of the compounds described in reference A is related to the inhibition of TNF production and the inhibitor of phosphodiesterase IV production. Therefore the invention described in reference A is not directly related to the use based on the inhibition of Cdk4 and/or Cdk6 according to the present invention.
In references B, C and D, substituted-2(1H)-pyridopyrazinone derivatives are disclosed. However, these compounds described in each reference are characterized in that they have a 3-indolyl group at position 3 of the pyrazinone ring, whereas the compounds according to the present invention are characterized in that they have a substituted phenyl or a substituted pyridyl which is structurally different from substituted 3-indolyl. Therefore the compounds according to the present invention are completely different in their chemical structure. Moreover, the use of the compounds described in references B, C and D is intended for cancer, inflammation, immune disorder, bronchopulmonary disorder, heart disease, and the like caused by the inhibition of proteinkinase C, so these inventions in references B, C and D are not directly related to the use based on the inhibition of Cdk4 and/or Cdk6 according to the present invention.
In reference E, heterocyclic compounds including substituted-2(1H)-pyridopyrazinone derivatives are disclosed. The compounds described in references E are however characterized in that they have a substituent other than hydrogen atom at position 3 of the pyrazinone ring, whereas the compounds according to the present invention are characterized in that the substituent at position 1 of the pyrazinone ring is fixed to hydrogen atom. Therefore the compounds according to the present invention are completely different in their chemical structure. Moreover, the use of the compounds described in reference E are intended for a factor Xa inhibitor, and thus the invention in reference E is not directly related to the use based on the inhibition of Cdk4 and/or Cdk6 according to the present invention.
In references F and G, heterocyclic compounds including substituted 2(1H)-pyridopyrazinone derivatives are disclosed. However such compounds described in each reference are characterized in that they have a substituent other than hydrogen atom in the first position of the pyrazinone ring, whereas the compounds according to the present invention are characterized in that the substituent in the first position of pyrazinone ring is fixed to hydrogen atom. Therefore the compounds according to the present invention are completely different in chemical structure from such known compounds. Moreover, the uses of the compounds described in these references are for electroluminescence element and pigment, so these inventions in reference F and G are different far from this invention in the art to which they belong.
Therefore, the present invention refers to a novel compound and its manufacturing procedure which are not described in any references yet, and is not be able to be easily achieved by person skilled in the art on the basis of the above-mentioned references of A to G.
As mentioned above, chromon derivatives are referred to as the compounds which have an inhibiting activity for Cdk family, but the inhibiting activity for Cdk4 is not so enough that the compounds which have still more high-inhibiting activity are needed.
Moreover, the novel compounds which have, additionally, heterogeneous inhibitory activity for Cdk6 and the like are needed.